| Summary: | In Gram-negative bacteria such as Escherichia coli, signal sequence-bearing secretory preproteins are targeted post-translationally from the cytosol to their final destinations. This mechanism is mainly performed by the ubiquitous Sec machinery, a multiprotein complex containing the molecular motor ATPase SecA, the secretion-dedicated chaperone SecB and a heterotrimeric protein-conducting channel consisting of the SecY, SecE and SecG subunits. Crystal structures have been obtained for rnonomeric, detergent-solubilised SecYEG in its 'closed' and SecA-bound states, revealing that the channel lies at the centre of a single protomer. However, many aspects of preprotein translocation remain uncertain, including the functional significance of the observation that in membranes SecYEG is predominantly dimeric. To address these uncertainties, total internal reflection fluorescence microscopy (TIRFM) was exploited to investigate the functional oligomeric state and monitor dynamics of the translocon at a single molecule level. This approach revealed that while monomers are sufficient for the SecA- and ATPdependent association of SecYEG with preproteins, active transport requires SecYEG dimers associated through the SecE subunit. In collaboration with the Collinson group (University of Bristol, UK), a molecular model of the functional translocon was proposed, rationalising the need for both SecYEG copies. The SecY channel is closed at the periplasmic side of the membrane by a small helical region termed the 'plug'. Relocation of the latter towards SecE during polypeptide translocation was investigated kinetically and spatially by ensemble and single molecule Forster resonance energy transfer (FRET), respectively. Intra-molecular conformational changes within SecA were also probed using FRET, the results suggesting that monomerisation of the SecA dimer occurs as a pre-activation step upon binding to SecYEG. Overall, the results presented in this Thesis describe the first use of single molecule imaging to study the bacterial Sec-translocon, and represent an integral part of the emerging applications of single molecule techniques in the membrane protein field.
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